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《Chinese Journal of Immunology》 2015-02
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Specificity of screening of short hairpin RNA targeting Mcl-1 gene in Raw264.7 cells

WANG Chan;WANG Xin-Min;WANG Fei-Yu;ZHANG Yu-Qing;CAO Xu-Dong;WU Jiang-Dong;WU Fang;ZHANG Wan-Jiang;ZHANG Le;Department of Microbiology Immunology,Department of Pathophysiology Medical School,Shihezi University/Laboratory of Xinjiang Endemic and Ethnic Diseases,Shihezi University;  
Objective: To transfect Mcl-1 shRNA into Raw264. 7 cells,and screen out specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene to figure out the effect of shRNA on Mcl-1 expression in murine macrophage cell line Raw264. 7. Methods: Specific shRNA was transfected into murine macrophage cell line Raw264. 7 via lipofectamine. Semi-quantitative RT-PCR and Western blot were respectively employed to test the changes in Mcl-1 mRNA level and Mcl-1 protein expressions 24 h and 48 h after the transfection,and the silencing effects of the three pairs of specific shRNA fragments corresponding to different sites were analyzed. Results: Specific shRNA fragments at 24 h and 48 h could effectively reduce Mcl-1 mRNA and protein level,with higher silencing effects than those of the normal group,the lipofectamine group and the negative control group. There were statistically significant differences among them( P 0. 05). Among the three pairs of specific shRNA fragments corresponding to different sites,Mcl-1 shRNA3 showed the most significant inhibiting effect on Mcl-1 mRNA and proteins. Conclusion: RNA interference can downregulate the level of Mcl-1 mRNA in murine macrophage cell line Raw264. 7 and greatly downregulate the expression of Mcl-1 protein. Specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene have been screened out successfully.
【Fund】: 国家自然科学基金资助项目(No.81260241);; 石河子大学项目(RCZX200922)
【CateGory Index】: R392
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